Metagenome analysis routinely uncovers a significant diversity of microorganisms with apparently-redundant functions in environmental and host-associated communities, bringing into question how such diversity is maintained as organisms compete for resources. The functional role of an organism in a community is routinely inferred by prediction of its genome-encoded metabolic functionality, which is highly sensitive to the quality and specificity of gene annotation but blind to how those genes are expressed. Global approaches can quantitatively identify changes in gene regulation with respect to changes in conditions, but do not shed light on the regulatory proteins responsible for their activation or repression nor the specific environmental signals they recognize.
Although global gene expression analyses (e.g., transcriptomics, proteomics) frequently implicates coordination of gene expression that is regulated by environmental conditions, identifying the specific mechanisms by which genes are regulated has been dependent on isolation of specific target microbes and genetic manipulation of cultured strains. Global approaches can quantitatively identify changes in gene regulation with respect to changes in conditions, but do not shed light on the regulatory proteins responsible for their activation or repression nor the specific environmental signals they recognize. Consequently, changes in expression of any given gene may stem from cascading, compensatory changes in gene regulation that are second- or third-order to the environmental stimulus. Validation of a putative regulatory protein initially identified using a global approach traditionally requires deletion of a putative regulator, examination of the effect on gene expression under various conditions, and DNA footprinting and/or gel-shift assays to identify the regulator's binding sites on the bacterial chromosome. This process has been dramatically quickened through chromatin immunoprecipitation (ChIP) hybridization or sequencing. In ChIP, antibodies against a known regulatory protein are used to precipitate DNA bound to the regulator, which is then sequenced to identify the protein's binding sites. Traditional ChIP-seq requires a priori knowledge of which regulators are important for processes of interest. Because ChIP is dependent upon antibody recognition of the target regulator, it entails either an arduous process of cloning the gene encoding a regulatory protein of interest, purifying the native regulatory protein, and generating antibodies or appending an epitope tag to a gene on the chromosome of a genetically-tractable host organism. These approaches are species-specific and technically challenging; consequently, ChIP-seq is typically employed in only well-studied and tractable organisms. Additionally, ChIP typically does not provide information regarding the signal to which a given regulator is responding as it binds DNA. As natural communities frequently contain diverse organisms that have not yet been cultivated, much less made genetically tractable, ChIP cannot be effectively used to dissect out the mechanisms by which multiple members respond to the identical environmental stimulus. Furthermore, for poorly-characterized axenic isolates, the amount of prior information and investment required to successfully perform ChIP against a putative regulator makes this approach highly investment-intensive and technically uncertain.
What is needed is an approach to gene regulation that is responsive to a specific small-molecule probe, is generalizable across species, and requires no prior knowledge and only modest investment. Such an approach would be applicable to examining gene regulatory mechanisms in both prokaryotic and eukaryotic organisms that respond to small molecule signals.